Localization of protein kinase C isoforms in the optic pathway of mouse embryos and their role in axon routing at the optic chiasm.

Protein kinase C (PKC) plays a key role in many receptor-mediated signaling pathways that regulate cell growth and development. However, its roles in guiding axon growth and guidance in developing neural pathways are largely unknown. To investigate possible functions of PKC in the growth and guidance of axons in the optic chiasm, we first determined the localization of major PKC isoforms in the retinofugal pathway of mouse embryos, at the stage when axons navigate through the midline. Results showed that PKC was expressed in isoform specific patterns in the pathway. PKC-α immunoreactivity was detected in the chiasm and the optic tract. PKC-ßΙΙ was strong in the optic stalk but was attenuated on axons in the diencephalon. Immunostaining for PKC-ε showed a colocalization in the chiasmatic neurons that express a surface antigen stage specific embryonic antigen-1 (SSEA-1). These chiasmatic neurons straddled the midline of the optic chiasm, and have been shown in earlier studies a role in regulation of axon growth and guidance. Expression levels of PKC-ßΙ, -δ and -γ were barely detectable in the pathway. Blocking of PKC signaling with Ro-32-0432, an inhibitor specific for PKC-α and -ß at nanomolar concentration, produced a dramatic reduction of ipsilateral axons from both nasal retina and temporal crescent. We conclude from these studies that PKC-α and -ßΙΙ are the predominant forms in the developing optic pathway, whereas PKC-ε is the major form in the chiasmatic neurons. Furthermore, PKC-α and -ßΙΙ are likely involved in signaling pathways triggered by inhibitory molecules at the midline that guide optic axons to the uncrossed pathway.

Heparan sulfate sugar modifications mediate the functions of slits and other factors needed for mouse forebrain commissure development.

Heparan sulfate proteoglycans are cell surface and secretory proteins that modulate intercellular signaling pathways including Slit/Robo and FGF/FGFR. The heparan sulfate sugar moieties on HSPGs are subject to extensive postsynthetic modification, generating enormous molecular complexity that has been postulated to provide increased diversity in the ability of individual cells to respond to specific signaling molecules. This diversity could help explain how a relatively small number of axon guidance molecules are able to instruct the extremely complex connectivity of the mammalian brain. Consistent with this hypothesis, we previously showed that mutant mice lacking the heparan sulfotransferases (Hsts) Hs2st or Hs6st1 display major axon guidance defects at the developing optic chiasm. Here we further explore the role of these Hsts at the optic chiasm and investigate their function in corpus callosum development. Each Hst is expressed in a distinct pattern and each mutant displays a specific spectrum of axon guidance defects. Particular Hs2st(-/-) and Hs6st1(-/-) phenotypes closely match those of Slit1(-/-) and Slit2(-/-) embryos respectively, suggesting possible functional relationships. To test functional interactions between Hs2st or Hs6st1 and Slits we examined optic chiasm and corpus callosum phenotypes in a panel of genotypes where Hs2st or Hs6st1 and Slit1 or Slit2 function were simultaneously reduced or absent. We find examples of Hs2st and Hs6st1 having epistatic, synergistic, and antagonistic genetic relationships with Slit1 and/or Slit2 depending on the context. At the corpus callosum we find that Hs6st1 has Slit-independent functions and our data indicate additional roles in FGF signaling.

Development of an in vivo adeno-associated virus-mediated siRNA approach to knockdown tyrosine hydroxylase in the lateral retrochiasmatic area of the ovine brain.

We developed a new technique of gene knockdown (KD) in a specific brain area of the ewe using an adeno-associated virus (AAV)-mediated short interfering RNA (siRNA) method to elucidate the importance of key factors of seasonal reproduction. Two 19-nucleotide sequences (TH1 or TH2) were chosen from the tyrosine hydroxylase (TH) gene. TH1, TH2 or a random sequence (TH3) was incorporated into an eGFP expressing AAV vector. Firstly, 5 microl of AAV-TH1 or AAV-TH2 solutions (8-9 x 10(11)Vg/ml) were stereotaxically injected into one A15 nucleus while the other received a control treatment. Ewes were killed after 15 or 75 days. The number of TH neurons was 49% and 36% lower on the AAV-TH1 treated side than on the control side 15 and 75 days post-injection, respectively. AAV-TH2 did not induce a significant variation in TH cell population. Finally, in order to increase the KD, two groups of ewes received 10 microl of AAV-TH1 either in a bolus injection or in two 5 microl inoculations carried out 2 weeks apart. Only ewes receiving a bolus injection showed a larger KD reaching 66% 2 months after inoculation. This method proved effective in reducing TH expression and will be further developed to understand cellular mechanisms driving seasonal functions.

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism.

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.

Protein tyrosine phosphatase-mu differentially regulates neurite outgrowth of nasal and temporal neurons in the retina.

Cell adhesion molecules play an important role in the development of the visual system. The receptor-type protein tyrosine phosphatase, PTPmu is a cell adhesion molecule that mediates cell aggregation and may signal in response to adhesion. PTPmu is expressed in the chick retina during development and promotes neurite outgrowth from retinal ganglion cell (RGC) axons in vitro (Burden-Gulley and Brady-Kalnay, 1999). The axons of RGC neurons form the optic nerve, which is the sole output from the retina to the optic tectum in the chick. In this study, we observed that PTPmu expression in RGC axons occurs as a step gradient, with temporal axons expressing the highest level of PTPmu. PTPmu expression in the optic tectum occurred as a smooth descending gradient from anterior to posterior regions during development. Because temporal RGC axons innervate anterior tectal regions, PTPmu may regulate the formation of topographic projections to the tectum. In agreement with this hypothesis, a differential response of RGC neurites to a PTPmu substrate was also observed: RGCs of temporal retina were unable to extend neurites on PTPmu compared with neurites of nasal retina. When given a choice between PTPmu and a second substrate, the growth cones of temporal neurites clustered at the PTPmu border and stalled, thus avoiding additional growth on the PTPmu substrate. In contrast, PTPmu was permissive for growth of nasal neurites. Finally, application of soluble PTPmu to retinal cultures resulted in the collapse of temporal but not nasal growth cones. Therefore, PTPmu may specifically signal to temporal RGC axons to cease their forward growth after reaching the anterior tectum, thus allowing for subsequent innervation of deeper tectal layers.

Effect of different illumination conditions and ionic environment on the guanylate cyclase activity in retina, optic nerve and optic chiasm of the rat.

Guanylate cyclase is sensitive to changes of light and dark periods in incubated extracts obtained from soluble fractions of the retina, optic nerve and optic chiasm. The changes in guanylate cyclase activity found, about 100-fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibits strongly the retinal guanylate cyclase activity, while it increases the activity of this enzyme in the optic nerve. A generalized photo-inhibited response of guanylate cyclase was observed in all studied tissues in animals adapted to the dark. This suggests that light could act as a double stimulus gating the central circuit which promotes the hydrolysis of cGMP via cGMP phosphodiesterase-rhodopsin-transducin cascade, and by direct inhibition of the retinal guanylate cyclase activity. Finally, different responses have been observed in the guanylate cyclase activity in relation with the ion exposure depending on the studied tissue. In summary, all indicate an important role for the soluble guanylate cyclase activity in retina, and other tissues involved in the visual process such as optic nerve and optic chiasm, which have not been examined until now.

Guanylate cyclase activity in retina optic nerve and optic chiasm of rats under different illumination conditions.

In the present work we have measured the guanylate cyclase activity in soluble fractions from several tissues relevant to the visual response under different illumination conditions. Guanylate cyclase was sensitive to changes of light/dark periods in incubated extract obtained from soluble fractions of retina, optic nerve and optic chiasm. The changes in soluble guanylate cyclase activity found, about 100 fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibit strongly the soluble retinal guanylate cyclase activity; while it increases the activity of this enzyme in the optic nerve. A generalized photoinhibited response of soluble guanylate cyclase was observed in all studied tissues in prolonged dark adapted animals. The effect of Na+ 1 and 10 mM, and free Ca++ 28 eta M and 2.8 microM on the guanylate cyclase activity was performed in the studied tissues. The enzymatic activity appeared to be inversely related in the retina and optic nerve with regard to the ion exposure, which may involve different ionic control mechanisms. All indicate an active role for the soluble guanylate cyclase in the phototransduction process not only in retina, also in other tissues relevant in the visual response.

Guanylate cyclase activity in retina optic nerve and optic chiasm of rats under different illumination conditions

In the present work we have measured the guanylase cyclase activity in soluble fractions from several tissues relevant to the visual response under different illumination conditions. Guanylate cyclase was sensitive to changes of light / dark periods in incubated extract obtained from soluble fractions of retina, optic nerve and optic chiasm. The changes in soluble guanylate cylcase activity found, about 100 fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibit strongly the soluble retinal guanylate cyclase activity; while it increases the activity of this enzyme in the optic nerve. A generalized photoinhibited response of soluble guanylate cyclase eas observed in all studied tissues in prolonged dark adapted animals. The effect of Na+ 1 and 10 nM, and free Ca++ 28 M and 2.8 MuM on the guanylate cyclase activity was performed in the studied tissues. The enzymatic activity appeared to be inversely related in the retina and optic nerve with regard to the ion exposue, which may involve different ionic control mechanisms. All indicate an active role for the soluble guanylate cyclase in the phototransduction process not only in retina, also in other tissues relevant in the visual response.

Guanylate cyclase activity in retina optic nerve and optic chiasm of rats under different illumination conditions

In the present work we have measured the guanylase cyclase activity in soluble fractions from several tissues relevant to the visual response under different illumination conditions. Guanylate cyclase was sensitive to changes of light / dark periods in incubated extract obtained from soluble fractions of retina, optic nerve and optic chiasm. The changes in soluble guanylate cylcase activity found, about 100 fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibit strongly the soluble retinal guanylate cyclase activity; while it increases the activity of this enzyme in the optic nerve. A generalized photoinhibited response of soluble guanylate cyclase eas observed in all studied tissues in prolonged dark adapted animals. The effect of Na+ 1 and 10 nM, and free Ca++ 28 M and 2.8 MuM on the guanylate cyclase activity was performed in the studied tissues. The enzymatic activity appeared to be inversely related in the retina and optic nerve with regard to the ion exposue, which may involve different ionic control mechanisms. All indicate an active role for the soluble guanylate cyclase in the phototransduction process not only in retina, also in other tissues relevant in the visual response. (AU)

Development of acetylcholinesterase activity in the lateral geniculate nucleus.

The pattern of acetylcholinesterase activity in the tree shrew (Tupaia belangeri) lateral geniculate nucleus (LGN) undergoes a number of striking changes during postnatal development. The adult tree shrew LGN is made up of six cellular layers divided by relatively cell-free interlaminar zones. At birth, however, the nucleus appears unlaminated when processed with conventional Nissl-staining techniques. The cellular lamination appears during the first postnatal week. The eyes open much later, typically at the end of the third week after birth. In the adult tree shrew, acetylcholinesterase (AChE) activity is found throughout the nucleus (both within and between the six cellular layers). In most sections examined, reaction product is slightly more intense in the lateral cell layers (4, 5, and 6). This is in sharp contrast to the pattern at birth (postnatal day zero, or P0). The detectable AChE activity at this age is apparently found in inchoate layers 1-2 and 4-5. Within these pairs, areas innervated by the ipsilateral eye (i.e., incipient layers 1 and 5) appear to contain more reaction product. From P0 to P4, the density of AChE activity increases in layers 1-2 and 4-5 and becomes detectable in the barely evident layers 3 and (usually) 6 at this age. By the middle of the second postnatal week, after laminae are clearly apparent with a Nissl stain, AChE activity has increased and is mainly associated with each cellular layer in the nucleus. During the third week after birth this pattern undergoes a radical shift. The most intense AChE activity is now in the interlaminar zones. Finally, as the adult pattern emerges, AChE activity increases in the cellular layers and all areas of the nucleus exhibit relatively high levels of AChE activity. Superimposed on this changing laminar pattern of AChE activity are changes related to the retinotopic map within the nucleus. Portions of the LGN representing central vision develop their characteristic pattern of activity several days ahead of the regions representing more peripheral visual field locations. AChE activity is also found transiently in the optic tract near the LGN during the first 3 postnatal weeks. Two (possibly three) groups of AChE-carrying fibers can be traced from the optic chiasm to their apparent sites of termination (or origin) in the parabigeminal nucleus, ventral lateral geniculate nucleus, and dorsal LGN. The activity present in the optic tract disappears shortly after eye opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential distribution of the 5-alpha-reductase in the central nervous system of the rat and the mouse: are the white matter structures of the brain target tissue for testosterone action?

In the brain of several animal species testosterone is converted into a series of 5-alpha-reduced metabolites, and especially into 17-beta-hydroxy-5-alpha-androstan-3-one (DHT), by the action of the enzyme 5-alpha-reductase. The formation of DHT has never been evaluated in the white matter structures of the brain, which are composed mainly of myelinated axons. The experiments here described were performed in order to study, in the rat and the mouse, the DHT forming activity of several white matter structures, in comparison with that of the cerebral cortex and of the hypothalamus. Two sampling techniques were used in the rat: microdissection under a stereo-microscope from frozen brain sections of fragments of corpus callosum, optic chiasm and cerebral cortex; fresh tissue macrodissection of subcortical white matter, cerebral cortex and hypothalamus. Only macrodissection was used in the mice. The data show that, independently from the sampling technique used, there are considerable quantitative differences in the distribution pattern of the 5-alpha-reductase activity within different brain structures. Both in the rat and in the mouse, the enzyme appears to be present in higher concentrations in the white matter structures, than in the cerebral cortex and in the hypothalamus. The present results clearly show that the subcortical white matter and the corpus callosum are at least three times as potent as the cerebral cortex in converting testosterone into DHT. An even higher 5-alpha-reductase activity has been found in the optic chiasm. Further work is needed in order to understand the possible physiological role of DHT formation in the white matter structures.

[Cholinergic mechanisms in the retina of rats]. / Cholinerge Mechanismen in der Retina bei Ratten.

In rats with extra- or intracranial dissection of the visual pathway the activities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were determined in retina, optic nerve and optic tract quantitatively by bio- and histochemical methods after one or five weeks of survival time. One week after intracranial (chiasma-near) dissection of the optic nerve an initial ipsilateral enhancement in the enzyme activity of AChE was found histochemically in retina layers and optic nerve. Five weeks following the dissection significant losses in AChE activity could be determined in the optic nerve, less pronounced in the retina. However, the activity of ChAc in the retina never was changed, neither after short nor after long survival times. Therefore, the high activity of both enzymes connected with cholinergic transmission normally present in different retinal layers must belong to local neurons, probably amacrine cells. To elucidate the question of the existence of a centrifugal cholinergic innervation of the retina one eye was enucleated. One week after operation a significant increase in the activity of AChE and ChAc was found in central parts of the dissected optic nerve and in the opposite optic tract indicating the existence of centrifugal cholinergic fibers in the optic nerve, but no evidence for the origin of these fibres could be obtained. The possible role of a central cholinergic control of the visual information processing already in retinal level is discussed in analogy to similar results obtained in the olfactory bulb.

Progressive somatofugal activation of cerebroside sulfotransferase in the developing chick optic fibers.

A myelination parameter of the chick optic pathway has been investigated: the activity of cerebroside sulfotransferase. Evidence is presented for a somatofugal progression of the enzyme activity peak along the optic nerve, chiasm, optic tract, tectum anterior, and tectum posterior. The enzyme activity appears on the fifteenth embryonic day and reaches a maximum in the nerve two days before hatching. One day after hatching, the peak is found in the chiasm, and three days after that in the optic tract. The peak is found in the tectum anterior on the tenth day. The results suggest a somatofugal progression of myelination.

Morphological and cytochemical study of a hypothalamochiasmatic perivascular neuronal system.

The topographical localization and some cytochemical characteristics of groups of perivascular neurons in the hypothalamus and in the optic chiasma have been studied. In the anterior hypothalamic area there are neuronal groups located around the blood vessels which penetrate the optic chiasma still associated with neurons. These perivascular neurons show some characteristics similar to those observed in neurosecretory hypothalamic neurons, but differ from them in that they show a greater intensity of thiaminopyrophosphatase and nucleoside-diphosphataseactivities. It is suggested that these neuronal groups may constitute a special hypothalamochiasmatic-perviscular-neurosecretory system (H. Ch. P. N. S.).

Topographical distribution of acetylcholinesterase and butyrylcholinesterase in the diencephalon and mesencephalon of the garden lizard (Calotes versicolor).

The present report incorporates the histochemical mapping of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) among the various nuclei and fiber tracts of the diencephalon and mesencephalon of Calotes veriscolor. The various nuclei, for both enzymes, present varying degrees of staining, ranging from negative nuclei, on the one hand, to mild and intense staining on the other hand. Almost all of the fiber tracts reveal intense activity in BChE preparations, while they demonstrate mild and moderate activities for AChE. The nature of the various nuclei in relation to enzymatic patterns is discussed.